The present invention relates to labeled reagents which are useful as detection reagents in analytical test systems. In particular, the present invention relates to enzyme-labeled antibodies which are useful as labeled reagents in an immunoassay.
The development of various immunoassay test formats and systems for the detection of analyte in a liquid test sample has given rise to the use of labeled reagents, particularly enzyme-labeled antibodies, to monitor the extent of antigen-antibody binding in an immunoassay. Generally, such test formats and systems involve a labeled reagent comprising a labeled form of the analyte of interest or an antibody thereto, which is combined with a test sample containing the analyte of interest to form a binding reaction system producing two species of the labeled reagent, a bound species and a free species. The relative amount or proportion of the labeled reagent that results in the bound species compared to the free species is a function of the presence or amount of analyte in the test sample. Where the labeled reagent is a labeled form of an antibody, such immunoassay is referred to as an immunometric assay or, more particularly, where the label of such labeled antibody is an enzyme, an enzyme immunometric assay.
The first use of enzyme-labeled antibodies was described by Avrameas and Uriel [Compt. Rend., Ser. D., Vol. 262, 2543-5(1966)] and Nakane and Pierce [J. Histochem. Cytochem., Vol. 14, 929(1966)] for use in immunocytochemical detection. Enzyme-labeled antibodies have also been used in quantitative enzyme-linked immunoassays [Engvall and Perlmann, Immunochemistry, Vol. 8, 871(1971) and U.S. Pat. No. 3,654,090], and since that time, various procedures have been described for coupling antibodies, particularly monovalent fragments [Fab', i.e., containing sulfhydryl groups in the hinge region, or Fab, i.e., containing no hinge region sulfhydryl groups] thereof, to various enzymes to provide enzyme-antibody conjugates of varying stoichiometries. Although divalent antibody fragments [F(ab').sub.2 ] or IgG can be employed in such assays, monovalent antibody fragments (Fab') are preferred because of improved assay sensitivity. Such improved sensitivity of Fab' fragments over F(ab').sub.2 fragments or IgG is due to the indistinguishability of monosaturated divalent antibodies from unsaturated antibodies in terms of their binding behavior, whereas when monovalent antibodies are employed, there will be a one-to-one correspondence between binding of sample analyte and displacement of labeled antibody.
For example, Freytag, et al. [Clin. Chem., Vol. 30, No. 3, 417-420 and Vol. 30, No. 11, 1809-1811(1984) and U.S. Pat. No. 4,434,236] describe methods for coupling Fab' monovalent antibody fragments to .sub..beta. -D-galactosidase. Similarly, Yoshitake et al. [Eur. J. Biochem., Vol. 101, 395-399(1979)] describe methods for coupling Fab' fragments to glucose oxidase.
The known methods of conjugation involve random techniques of protein-protein conjugation which result in conjugates of varying stoichiometry, as well as contaminants such as free enzyme and free antibody components. Such contaminants, when present, significantly interfere with the sensitivity of an enzyme immunoassay. In particular, free enzyme in the conjugate mixture results in an increased background in an immunometric assay and, accordingly, decreased precision. Similarly, the presence of free antibody or fragments thereof in a conjugate preparation results in decreased assay sensitivity and nonlinear dose response in an immunometric assay. Moreover, in order to obtain high assay sensitivity and a linear dose response, it is preferred to employ monoconjugates (i.e., one enzyme component coupled to a single antibody component) rather than polyconjugates (i.e., enzyme component coupled to more than one of the antibody component) in an immunoassay.
Accordingly, a number of methods for conjugating enzymes and antibodies to achieve a monoconjugate and methods of purification in order to overcome the problems presented by the presence of enzyme and/or antibody contaminated conjugates have been attempted. However, the preparation of a substantially pure enzyme-antibody monoconjugate has been successful only with enzymes having a comparable molecular weight to the antibody component. For example, Ishikawa, et al. [J. of Applied Biochemistry, Vol. 4, 41 (1982) and J. Biochem., Vol. 92, 1413(1982)] describe horseradish peroxidase (activated with succinimidyl-4-(N-maleimido-methyl)cyclohexane-1-carboxylate) coupled to Fab' fragments where the monoconjugate thereof was isolated by gel filtration and immunopurified [Analytical Letters, , Vol. 17, 229 and 2076(1984)].
Although gel filtration and immunopurification have been shown to be successful for isolating Fab'-horseradish peroxidase monoconjugates as heretofore described, such methods are inadequate to separate and isolate monoconjugates of Fab' fragments and enzymes of disparate molecular weights, such as Fab'-glucose oxidase [Eur. J. Biochem., Vol. 101, 395(1979)], Fab'-alkaline phosphatase [Scand. J. Immunol., pk Vol. 8, 43-55(1980)], and Fab'-.sub..beta. -D-galactosidase [Analytical Letters, Vol. 18(1311), 1331-1334(1985)]. Such methods are not only inadequate to separate monoconjugates from polyconjugates, as well as from free enzyme components, immunopurification often results in low conjugate recovery and risk of enzyme and/or antibody inactivation.
There is also described in FEBS Letters, Vol. 90, 51(1978), a gel filtration-elution method to isolate IgG-invertase monoconjugate by immobilizing invertase to a Con A column, activating the immobilized invertase, and then adding IgG to the column. The resulting conjugate was then desorbed from the affinity support and purified by gel filtration. However, the method produced conjugates having protein:enzyme and antibody binding site:enzyme ratios of 1:1 and 2:1, respectively, and, accordingly, did not yield a monoconjugate, i.e., comprising 1:1 proportions for both protein:enzyme and antibody binding site:enzyme ratios. In addition, such method resulted in a major portion of unconjugated IgG, i.e., low levels of conjugation, and further, the gel filtration step would be effective to isolate a monoconjugate only if the two proteins were comparable in molecular weight as previously discussed.
The difficulty of separating and isolating monoconjugates of antibodies and enzymes of disparate molecular weights to obtain a pure preparation thereof has also been illustrated [BioChromatography, Vol. 1, 42(1986)] employing the most advanced HPGFC (high performance gel-filtration chromatography) techniques, particularly IgG conjugates of horseradish peroxidase, alkaline phosphatase, and .beta.-D-galactosidase. In particular, an IgG-.beta.-D-galactosidase conjugate mixture was purified on a gel filtration column (Sepharose.RTM. CL-4B) to obtain a fraction consisting predominantly of free .beta.-D-galactosidase, and other large molecular weight aggregates. This pooled fraction was loaded to the HPGFC column and three peaks without baseline resolution were observed in the chromatogram. The predominant peak was unreacted .beta.-D-galactosidase; another peak which eluted first in the chromatogram was presumably aggregated conjugate. The small peak which eluted between these two was alleged to be monoconjugate (1:1 IgG-.beta.-D-galactosidase) although no evidence was offered. Undoubtedly this fraction contained some monoconjugate but was probably contaminated with both free enzyme and polyconjugates. Given this general lack of resolution in this molecular weight range, the likelihood of resolving a 1:1 Fab-.beta.-D-galactosidase conjugate would be very small using this technique.
Accordingly, it is an object of the present invention to provide a method for separating and isolating enzyme-antibody monoconjugates and polyconjugates comprising enzyme and antibody components of disparate molecular weights from each other and from contaminating free enzyme components and free antibody components.
Another object of the present invention is to permit the use of enzyme and antibody stoichiometries which drive the coupling reaction of enzyme and antibody to maximum conjugate production at high, preparative yields.
Further, it is an object of the present invention to provide a highly sensitive and precise immunometric assay having little or no background signal.
Still further, it is an object of the present invention to provide an enzyme-antibody conjugate having maximum enzyme and antibody activity.